Abstract
We previously reported when a portion of the Requiem (REQ/DPF2) messenger ribonucleic acid (mRNA) 3’ untranslated region (3’UTR), referred to as G8, was overexpressed in K562 cells, β-globin expression was induced, suggesting that the 3’UTR of REQ mRNA plays a physiological role (Kim et al., 2014). To identify trans-acting factors that bind to the REQ 3’UTR, we describe the RNA ligand based cDNA expression library screening method. This protocol could be adapted to detect specific RNA-protein interactions. Following this method, we identified six positive clones in the initial round of screening and four pure clones after sib-screening. This protocol was originally published in Kim et al. (2014).
Keywords: RNA-protein interaction, Phage display, Requiem (REQ/DPF2), CDNA library screening
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. Scheme of RNA-Ligand based cDNA expression library screening Figure 2. Autoradiograph showing positive putative clones after primary screening. All RNA-binding proteins were additionally confirmed by secondary/tertiary screening.
Notes
Figure 3. Autoradiography showing high background due to the low concentration of competitor yeast RNA (step E) and poor washing of non-specific bound radioactivity (step F)
Recipes
Acknowledgments
This work was supported by Basic Science Research Program (2010-00252250 to C. G. K.), National Research Foundation (NRF), Ministry of Education, Science and Technology (MEST), Republic of Korea. Converging Research Center Program (2013K000283 to C. G. K.), Ministry of Science, ICT & Future Planning (MSIFP), Republic of Korea.
References
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